Summary

Mammalian motor circuits control voluntary movements by transmitting signals from the central nervous system (CNS) to muscle targets. To form these circuits, motor neurons (MNs) must extend their axons out of the CNS. Although exit from the CNS is an indispensable phase of motor axon pathfinding, the underlying molecular mechanisms remain obscure. Here, we present the first identification of a genetic pathway that regulates motor axon exit from the vertebrate spinal cord, utilizing spinal accessory motor neurons (SACMNs) as a model system. SACMNs are a homogeneous population of spinal MNs with axons that leave the CNS through a discrete lateral exit point (LEP) and can be visualized by the expression of the cell surface protein BEN. We show that the homeodomain transcription factor Nkx2.9 is selectively required for SACMN axon exit and identify the Robo2 guidance receptor as a likely downstream effector of Nkx2.9; loss of Nkx2.9 leads to a reduction in Robo2 mRNA and protein within SACMNs and SACMN axons fail to exit the spinal cord in Robo2-deficient mice. Consistent with short-range interactions between Robo2 and Slit ligands regulating SACMN axon exit, Robo2-expressing SACMN axons normally navigate through LEP-associated Slits as they emerge from the spinal cord, and fail to exit in Slit-deficient mice. Our studies support the view that Nkx2.9 controls SACMN axon exit from the mammalian spinal cord by regulating Robo-Slit signaling.

Our observation that the homeodomain TF Nkx2.9 is likely to be required for SACMN axons to leave the CNS (Dillon et al., 2005) prompted us to further characterize the role of Nkx2.9 in motor axon exit. Here we show that, in mice lacking Nkx2.9, SACMN axons appropriately project to the LEP but assemble into an ectopic longitudinally projecting SAN within the spinal cord. We also identify the axon guidance receptor roundabout 2 (Robo2) (Ypsilanti et al., 2010) as a likely downstream effector of Nkx2.9 by showing that Robo2 expression in SACMNs is downregulated in Nkx2.9 null mice and that SACMN axons fail to exit the spinal cord in Robo2-deficient animals. Furthermore, the Robo2 ligands Slit1-3 are present at the LEP, SACMN axons fail to exit the CNS in Slit null mice, and Slit promotes SACMN axon outgrowth in vitro. Collectively, our findings are consistent with Nkx2.9 controlling SACMN axon exit from the CNS by regulating Robo2-Slit interactions at the LEP.

In Nkx2.9–/– embryos, SACMN axons fail to exit the CNS and assemble into an ectopic SAN within the spinal cord. (A-H) Wild-type (WT) (A,B, E10.5; E,F, E11.5) and Nkx2.9–/– (C,D, E10.5; G,H, E11.5) embryos were labeled with either anti-BEN and anti-laminin (A-D) or anti-BEN and anti-L1 (E-H). (A,B) In E10.5 WT embryos, BEN-labeled spinal accessory motor neuron (SACMN) axons extend through the lateral exit point (LEP) and assemble into a spinal accessory nerve (SAN) located outside the CNS. (C,D) SACMN axons appropriately extend to the LEP in Nkx2.9–/– mice but fail to exit and remain within the anti-laminin-labeled margin of the spinal cord (arrow), where they appear to assemble into an ectopic SAN (arrowhead) (n>5). B and D are magnified views of the boxed areas in A and C, respectively. (E-H) A BEN/L1-expressing SAN (arrowhead) forms outside of the spinal cord and adjacent to the LEP (arrow) in WT embryos, whereas in Nkx2.9–/– mice the SAN inappropriately forms within the spinal cord adjacent to the LEP and BEN-expressing SACMN (H, asterisks). F and H are magnified views of the boxed areas in E and G, respectively. (I) The distance from the base of the floor plate (FP) to the SAN (see upper schematic) was not statistically different in WT (dark gray; SAN) as compared with Nkx2.9–/– (light gray; ectopic SAN) embryos (n=4). Error bars indicate s.d. (J) Schematized open-book (OB) preparation from cervical levels (C1-C4) of the spinal cord. RP, roof plate. (K-N) OB preparations derived from an E11.5 Nkx2.9 null embryo and a WT littermate labeled with anti-BEN. In contrast to WT embryos (K,L), an ectopic SAN is located within the spinal cord of Nkx2.9–/– embryos (M,N; n=3). L and N are magnified views of the boxed areas in K and M, respectively. (O) The trajectory of SACMN axons in WT and Nkx2.9–/– embryos. Scale bars: 50 μm in C for A,C; 25 μm in D for B,D; 100 μm in G for E,G, in K for K,M, in L for L,N; 5 μm in H for F,H.

The Qiagen RNeasy Micro Kit was used to isolate total RNA from E11.5 cervical spinal cords (excluding dorsal spinal cord and floor plate; WT, n=3; Nkx2.9–/–, n=3). RNA was amplified using the WT-Ovation Pico RNA Amplification Kit (NuGEN Technologies), followed by hybridization to GeneChip Mouse Genome 430 2.0 Array (Affymetrix) by the Albert Einstein College of Medicine Affymetrix facility. Results from three independent arrays were averaged. The raw data (CEL files) from each probe set were normalized by RMA methods using GeneSpring GX 10.0 software (Agilent). The log2 transformed signal intensities were averaged for biological replicates and the mean value was used to compute fold change. Differentially expressed genes were identified as those exhibiting a fold change exceeding 1.5, as several previous studies have used this cut-off (Hughes et al., 2000; Schachter et al., 2002; Wang et al., 2003; Yuan et al., 2005) and a fold change of less than 2 can be biologically significant (Hughes et al., 2000).

Appropriate numbers of properly specified SACMNs are formed in Nkx2.9-deficient mice. (A) Schematic of SACMN and ventral motor neuron (vMN) development in the ventral spinal cord. SACMNs (green circle) and vMNs (red circle) arise from MN progenitors, which migrate away from the ventricular zone to settle in dorsolateral or ventrolateral regions, respectively, of the spinal cord. (B-G) Cryosections derived from WT (B-E, E10.5; F,G, E11) embryos labeled with various SACMN or vMN markers show that SACMNs and their axons (arrow) migrate through vMNs [SACMN (v)] and SACMNs that have settled near the LEP [SACMN (d)] and express BEN, Islet1 and Phox2b, whereas vMNs express Islet1 and HB9. C, E and G are magnified views of the boxed areas in B, D and F, respectively. (H-K) E11.5 WT (H,I) and Nkx2.9–/– (J,K) embryos were labeled with anti-Phox2b (H,J) and colabeled with anti-Phox2b and anti-BEN (I,K). I and K are magnified views of the boxed areas in H and J, respectively. (L,M) The number of Phox2b+ BEN+ (E11.5; n=4) and Islet1+ BEN+ (E10.5; n=3) SACMNs is not statistically different between Nkx2.9–/– and WT/heterozygote littermates. Error bars indicate s.d. Scale bars: 50 μm in F for B,D,F, in J for H,J; 25 μm in G for C,E,G, in K for I,K.

Photodocumentation, quantification and statistical analyses

Images were captured and processed as described (Dillon et al., 2005). Confocal images were acquired on a FluoView 500 confocal microscope (Olympus). ImageJ64 software was used to perform measurements. The Mann-Whitney test was performed for all statistical analyses using GraphPad Prism software (version 5.0). Cell body and nerve counts utilized at least five cryosections per animal per genotype. Axon outgrowth was assessed by converting grayscale into binary images using ImageJ64 and outlining areas of equivalent size located between the margins of SACMN axon-containing spinal cord explants and the aggregates of either mock- or Slit2-treated HEK293 cells. The percentage of the delimited area occupied by BEN-positive staining was measured using ImageJ64.

RESULTS

In Nkx2.9–/– mice, SACMN axons fail to exit the spinal cord and inappropriately assemble into an ectopic SAN

We previously reported that SACMN axons apparently fail to exit the CNS in Nkx2.9 null mice (Dillon et al., 2005). To unambiguously determine whether SACMN axons remain confined to the spinal cord in these mice, we labeled transverse cryosections from the cervical spinal cord of E10.5 Nkx2.9–/– embryos and their WT littermates with anti-BEN, as a selective marker of SACMNs, and anti-laminin, which demarcates the margin of the spinal cord (Fig. 1A-D). BEN-expressing SACMN axons failed to project across the laminin border in Nkx2.9–/– mice (Fig. 1C,D). This does not appear to reflect a developmental delay as SACMN axons do not exit the spinal cord in Nkx2.9–/– mice analyzed as late as E13.5, well after SACMN axons normally assemble into the SAN (Dillon et al., 2005) (data not shown).

Robo2 expression is reduced in Nkx2.9–/– mice. (A-F) E10.5 cryosections derived from WT (A,C,E) or Nkx2.9–/– (B,D,F) mice were subjected to in vitro hybridization for BEN (Alcam) (A,B) or Robo2 (C,D) mRNA, or labeled with anti-BEN and anti-Robo2 (E,F). Similar levels of BEN mRNA are expressed by SACMNs (black arrowheads) in WT (A) and Nkx2.9–/– (B) embryos (n>3). SACMN-associated Robo2 mRNA is reduced in Nkx2.9–/– embryos (D; n=3) as compared with WT littermates (C). In Nkx2.9–/– embryos, SACMN axons (arrow) and the ectopic SAN (white arrowhead) display reduced levels of Robo2 protein (F; n=3) compared with WT littermates (E). The dotted line indicates the margin of the spinal cord. Scale bars: 100 μm in D for A-D; 25 μm in F for E,F.

We next asked whether SACMN axons assemble into an ectopic SAN within the spinal cord of Nkx2.9 null mice by labeling transverse cryosections from E11.5 embryos with anti-BEN and anti-L1 (L1cam – Mouse Genome Informatics), which labels longitudinally projecting spinal axons (Imondi et al., 2000). A BEN/L1-labeled nerve was located adjacent to the LEP but within the spinal cord in Nkx2.9–/– mice (Fig. 1G arrow, 1H arrowhead). To determine whether this ectopic nerve projects along the anterior-posterior (A-P) extent of the cervical spinal cord, we analyzed OB preparations derived from E11.5 Nkx2.9–/– and Nkx2.9+/+ embryos. Consistent with OB preparations lacking an external SAN (Fig. 1J), a BEN-positive SAN was only observed within the spinal cord of Nkx2.9–/– mice (Fig. 1K-O). Together, these findings indicate that Nkx2.9 is selectively required for SACMN axon exit. By contrast, vMN axons appropriately emerge from the spinal cord in Nkx2.9–/– mice (supplementary material Fig. S1).

Appropriate numbers of SACMNs, which likely arise from Nkx2.9+ progenitors, form in Nkx2.9 null mice

To rule out the possibility that the lack of SACMN axon exit in Nkx2.9–/– embryos reflects a reduction in SACMN number, we first identified a panel of SACMN markers by labeling cryosections from the cervical spinal cord of WT embryos with anti-BEN and antibodies specific for the MN-associated TFs Islet1 (Ericson et al., 1992; Pfaff et al., 1996), HB9 (Mnx1 – Mouse Genome Informatics) (Arber et al., 1999) or Phox2b (Pattyn et al., 2000). At E10.5-11, BEN-positive SACMNs express the generic MN marker Islet1 (Fig. 2A-C) and the branchiomotor neuron marker Phox2b (Hirsch et al., 2007) (Fig. 2A,F,G), but not HB9 (Fig. 2A,D,E). Therefore, we analyzed the numbers of BEN+ Phox2b+ and BEN+ Islet1+ SACMNs in Nkx2.9 null and WT littermates and found that appropriate numbers of properly specified SACMNs are generated in the absence of Nkx2.9 (Fig. 2H-M).

To determine whether Nkx2.9 functions cell-autonomously to facilitate SACMN axon exit we compared the distribution of lacZ-expressing cells and SACMNs within the spinal cord of a Nkx2.9-lacZ knock-in mouse line (Tian et al., 2006). In E9.5 embryos, anti-β-galactosidase staining overlapped with Nkx2.9 mRNA above the floor plate (FP), suggesting that Nkx2.9+ progenitors give rise to lacZ-positive cells (supplementary material Fig. S2A-C). At E10.5, X-Gal staining of sections from Nkx2.9-lacZ–/– embryos identified strong labeling above and adjacent to the FP and within cells (presumably SACMNs) that migrate dorsolaterally towards the LEP (supplementary material Fig. S2D). To investigate the possibility that Nkx2.9 is required to maintain the identity of these lacZ-positive cells, we labeled cryosections derived from E10.5 Nkx2.9-lacZ+/– and Nkx2.9-lacZ–/– embryos with anti-β-galactosidase and antibodies to the SACMN markers BEN, Islet1 or Phox2b. β-galactosidase-expressing neurons were labeled by each of these markers (supplementary material Fig. S2E-X), and a subset of β-galactosidase-positive cells within the ventricular zone, which were likely to be Nkx2.9+ progenitors, expressed Phox2b (supplementary material Fig. S2S,U,V,X, asterisk). These observations are consistent with SACMNs arising from a subset of Nkx2.9+ progenitors and indicate that the specification/identity of SACMNs is unaltered in Nkx2.9–/– mice.

SACMN axons fail to exit the spinal cord in Robo2-deficient mice. (A-T) Cryosections derived from E10.5 Robo2+/+ (A,B), Robo2–/– (F,G), Robo1+/+ (K,L) and Robo1–/– (P,Q) embryos were labeled with anti-laminin and anti-BEN (first column) or anti-NF alone (second column). E11.5 Robo2+/+ (C-E), Robo2–/– (H-J), Robo1+/+ (M-O) and Robo1–/– (R-T) embryos were colabeled with anti-BEN (third column) and anti-NF (fourth column). BEN/NF-expressing SACMN axons fail to exit the spinal cord and form an ectopic SAN (asterisk) within the CNS in Robo2–/– mice (H-J), whereas in Robo1–/– mice (R-T), just as in Robo2+/+ (C-E) and Robo1+/+ (M-O) embryos, a SAN (A) formed outside the CNS. As in E10.5 Robo2–/– embryos, the majority of BEN/NF-expressing SACMN axons fail to exit the spinal cord at E11.5. At E11.5, several small ectopic SANs (asterisks) form within the spinal cord (H-J), as compared with WT mice (C-E). A small number of SACMN axons emerge from the CNS and form an external SAN at E11.5 (H-J, arrowhead). The bar charts show that the average number of external SANs per section is reduced in E10.5 Robo2–/– mice (top; n=4), but unaltered in Robo1–/– mice (bottom; n=3), as compared with WT mice. Error bars indicate s.d. SAN formation is unperturbed in E11.5 Robo1–/– mice (R-T; n=2), as compared with WT littermates (M-O; n=2). Scale bar: 50 μm.

We also carried out an unbiased microarray screen to identify genes dysregulated in Nkx2.9–/– embryos (supplementary material Table S1) by comparing total RNA isolated from the ventral half of the cervical spinal cord of E11.5 WT and Nkx2.9–/– embryos. This revealed that Robo2 mRNA levels were ∼2-fold lower in Nkx2.9-deficient mice than in WT mice (supplementary material Table S1).

Given these findings, we focused our subsequent studies on investigating the regulation of Robo2 expression in Nkx2.9–/– embryos and the role of Robo2 in SACMN axon exit.

Robo2 is expressed by SACMNs and is downregulated in Nkx2.9 null mice

To validate the results of our screens, we labeled cryosections derived from E10.5 Nkx2.9–/– embryos and their WT littermates with a Robo2 riboprobe or anti-Robo2. In WT embryos, BEN-expressing SACMNs express Robo2 mRNA (Fig. 3A,C, black arrowheads) and Robo2 protein is expressed on SACMN axons (Fig. 3E, arrows) and on the SAN (Fig. 3E, white arrowheads). In Nkx2.9–/– embryos, SACMN-associated Robo2 mRNA levels are reduced (Fig. 3B,D, black arrowheads) and Robo2 protein is downregulated on SACMN axons (Fig. 3F, arrows) that fail to exit the spinal cord, as well as on the ectopic SAN (Fig. 3F, white arrowheads). These observations are consistent with Robo2 operating as a downstream effector of Nkx2.9.

SACMN axons do not exit the spinal cord in Robo2-deficient mice

To determine whether Robo2 is required for SACMN axon exit, we labeled E10.5 cryosections derived from Robo2–/– mice and their WT littermates with anti-BEN and anti-laminin or anti-neurofilament (NF) (Fig. 4A-J). Phenocopying Nkx2.9 null mice, the majority of SACMN axons appropriately projected to the LEP but failed to exit the CNS in E10.5 Robo2–/– animals (Fig. 4F,G, asterisks), and most SACMN axons remained confined to the spinal cord at E11.5 (Fig. 4H-J). Since a small subset of SACMN axons exited the spinal cord in Robo2–/– mice (Fig. 4H-J, arrowheads), we quantified the presence or absence of an external SAN (SACMN axon-containing nerve bundle) on both sides of the spinal cord in WT and Robo2–/– embryos. Consistent with the failure of most SACMN axons to exit the CNS, there was a significant reduction in the mean number of external SANs in Robo2–/– mice (Fig. 4, top bar chart). Although Robo1 mRNA is expressed by SACMNs and Robo1 and Robo3 protein is present on the SAN (data not shown), SACMN axon pathfinding/exit is not perturbed in Robo1 (Fig. 4K-T, bottom bar chart) and Robo3 (data not shown) null embryos. Accordingly, Robo2 is the sole Robo receptor required for SACMN axon exit from the spinal cord.

Slits are expressed at the LEP and SACMN axon exit is perturbed in Slit null mice

Since SACMN axons appropriately project away from the FP and towards the LEP but fail to exit the spinal cord in Robo2–/– mice, short-range interactions between Robo2 and its Slit ligands at the LEP might normally facilitate SACMN axon exit. To test this hypothesis, we labeled serial cryosections derived from E10.5 WT embryos with Slit1, Slit2 or Slit3 riboprobes (Fig. 5A-D). High levels of Slit2 and lower levels of Slit1 and Slit3 are present at the LEP, and each Slit gene is expressed in vMNs and the FP. Based on these observations, we analyzed SACMN axon pathfinding in mice lacking one or more Slits by labeling cryosections from E10.5 Slit1–/–, Slit2–/– or Slit1–/– Slit2–/– embryos with anti-BEN and anti-NF. Whereas SACMN axon pathfinding/exit was not perturbed in the absence of Slit1 (Fig. 6D-F′) or Slit2 (Fig. 6G-I′), SACMN axons failed to exit the spinal cord in mice lacking both Slit1 and Slit2 (Fig. 6J-L′). In the double-mutant mice, SACMN axons formed more than one ectopic SAN within the spinal cord, just as they do in Robo2–/– mice (Fig. 6J-L′, asterisks). The number of ectopic nerve bundles was significantly increased, and there was a corresponding reduction in the number of normally positioned external SANs, in Slit1–/– Slit2–/– embryos as compared with WT mice and mutant mice lacking other combinations of Slits (Fig. 6M,N). These findings identify Slit1 and Slit2 as the LEP-associated Robo2 ligands required for SACMN axon exit, and suggest that the increase in the number of ectopic nerves within Slit1–/– Slit2–/– mice might arise from defasciculation of longitudinally projecting SACMN axons.

Given that multiple ectopic SANs appear to form in Robo2–/– and Slit1–/– Slit2–/– mice, we asked whether these nerves appropriately project along the A-P axis of the spinal cord by labeling OB preparations derived from the cervical spinal cord of these mice with anti-BEN (Fig. 9A-D). These analyses revealed that SACMN axons form disorganized nerve fascicles along the A-P axis of the spinal cord in mice lacking either Robo2 or Slit1/2 (Fig. 9C,D, arrows), supporting a role for Robo2-Slit interactions in the proper growth of longitudinally projecting SACMN axons.

Nkx2.9 regulates Robo2 expression

Nkx genes encode homeodomain-containing TFs that regulate cell type specification and organogenesis (Briscoe et al., 2000; Briscoe et al., 1999; Harvey, 1996; Stanfel et al., 2005), and Nkx2.9 is required for FP development and commissural axon guidance (Holz et al., 2010). We show that SACMN-associated Robo2 mRNA and protein are reduced in Nkx2.9–/– embryos, identifying Robo2 as the first candidate CNS-associated downstream effector of any vertebrate Nkx gene. By contrast, we observed no changes in Slit mRNA expression in these animals (data not shown). Although the presence of putative Nkx2 binding sites within the Robo2 promoter (data not shown) is consistent with Robo2 being a direct target of Nkx2.9, forced expression of Nkx2.9 does not modulate Robo2 expression (supplementary material Fig. S3).

Robo2 and Slit1/2 are required for SACMN axon exit

Our findings are the first to show that Robo2-Slit signaling is required to facilitate motor axon exit from the spinal cord. Since Slit2 appears to be the most abundant LEP-associated Slit, it was surprising to find that SACMN axons appropriately exit the spinal cord in mice lacking Slit2. Possible explanations for these observations are that low levels of Slit1 are capable of facilitating SACMN axon exit and/or that Slit1 and Slit2 operate redundantly to regulate exit. The finding that SACMN axons do not exit the spinal cord in Slit1–/– Slit2–/– mice further indicates that LEP-associated Slit3 does not promote SACMN axon exit on its own. Nevertheless, in the absence of published data indicating that Robo2 binds each of the vertebrate Slits with different affinities, it is difficult to reconcile why Slit1, but not Slit3, appears to compensate for the loss of Slit2. In contrast to our loss-of-function data, misexpression of Slits in the chick spinal cord does not perturb SACMN axon pathfinding (supplementary material Fig. S4).

A model for the dynamic role of Robo2-Slit interactions in regulating SACMN axon pathfinding towards and through the LEP. (A,B) In WT mice (A, left), Robo2-expressing (Robo2) SACMN axons (green) navigate in close proximity to three sources of Slits (red circles) as they extend away from the FP (1), grow through vMNs (2), and project to and through the LEP (3). SACMNs express Robo2 and Slits as they pathfind away from the FP and traverse vMNs, and through potential cis attenuation of Robo2-Slit signaling their axons become insensitive to FP- and vMN-derived Slits (1, 2). SACMN axons are normally repelled by the FP-derived chemorepellent Netrin (–) and could be guided to the LEP by exit point-derived chemoattractants (+). Once Robo2-positive SACMN axons reach the LEP, they no longer express Slits, become responsive to LEP-associated Slits (3), and attractive Robo2-Slit interactions facilitate their exit from the spinal cord. By contrast, SACMN axons in Nkx2.9–/– (dark gray, Robo2↓, A right), Robo2–/– (light gray, Robo2, B left) and Slit1–/– Slit2–/– (green, Robo2, B right) mice appropriately reach the LEP but fail to exit the spinal cord. (C) Model for Nkx2.9 control of SACMN axon exit via Robo-Slit signaling. (I) Nkx2.9 regulates Robo2 expression in SACMNs. (II) Attractive short-range Robo2-Slit interactions at the LEP facilitate SACMN axon exit from the CNS.

The dynamic role of Robo-Slit interactions in SACMN axon pathfinding

Given that the Slit-rich FP and ventral spinal cord would be expected to represent repulsive territories for Robo2-expressing SACMN axons, how are these axons capable of growing away from the FP and through the ventral spinal cord (Fig. 10A)? As is the case for vMNs (Bai et al., 2011; Brose et al., 1999), SACMNs are likely to co-express Robo2 and Slits as their axons navigate these initial segments of their trajectory (Figs 7, 10). Thus, Slits produced by SACMN cell bodies might occupy SACMN axon-associated Robo receptors, rendering them insensitive to FP- and ventral spinal cord-associated Slits, analogous to the co-expression of Slit and Robo in vMNs inhibiting the responsiveness of their axons to Netrin (Bai et al., 2011). Similarly, co-expression of Sema3A and Npn1 on vMNs prevents their axons from responding to exogenous Sema3A (Moret et al., 2007), and the attenuation of Eph receptor signaling via ephrins in cis alters the responsiveness of vMN axons to trans ephrins (Kao and Kania, 2011).

Our observations suggest that short-range, presumably attractive/positive (Fig. 8) interactions between Robo2-expressing SACMN axons and LEP-associated Slits promote SACMN axon exit from the spinal cord. Consistent with this possibility, mesodermal cells (Kramer et al., 2001) and tracheal branches (Englund et al., 2002) are attracted to Slit in Drosophila embryos, and human leukocytes are attracted to a Slit source in vitro (Ye et al., 2010). Drosophila mesodermal cells are also capable of responding to Slit as both an attractant and repellent during sequential phases of their migration (Kramer et al., 2001). By analogy, SACMN axons might alter their responsiveness to LEP-associated Slits, first perceiving Slits as attractants and then as repellents, and this would ultimately push SACMN axons out of the spinal cord. We favor the possibility that Slits produced by LEP-associated cells facilitate SACMN axon exit; however, Robo2-expressing SACMN axons could transport ventral midline-associated Slits to the LEP, just as the Frazzled receptor captures and redistributes its Netrin ligand to modulate axon pathfinding within the Drosophila CNS (Hiramoto et al., 2000). Selectively eliminating Slits from LEP-associated cells would clarify which sources of Slits are required for SACMN axon exit.

To ultimately emerge from the spinal cord, motor axons must break through the basement membrane surrounding the neural tube. Although our studies do not directly address this aspect of SACMN axon pathfinding, it seems plausible that Robo2-Slit interactions promote cytoskeletal remodeling, which might alter the shape of SACMN growth cones and facilitate exit. Consistent with this possibility, the complexity of SACMN axon-associated growth cones is dramatically reduced as they exit the LEP (Snider and Palavali, 1990), and this might facilitate their passage through small ‘gaps’ formed by glial end-feet at motor exit points (Fraher et al., 2007).

Alternatively, Slit activation of Robo2 at the LEP might trigger the release of either soluble or membrane-bound proteolytic enzymes such as matrix metalloproteinases (MMPs) (McFarlane, 2003), which are capable of breaking down the basal lamina and potentially promoting SACMN axon exit. Our microarray results indicate that the expression of the ADAM metallopeptidase Adamts3 is reduced in Nkx2.9–/– mice, raising the possibility that MMPs might regulate SACMN axon exit (supplementary material Table S1). In one scenario, SACMN axons may extend small, localized protrusions termed invadopodia that selectively secrete MMPs, which degrade the basement membrane (Bravo-Ambrosio and Kaprielian, 2011). No matter how SACMN axons break through the border between the CNS and the peripheral nervous system, we have provided evidence that short-range Robo2-Slit interactions are likely to regulate this crucial phase of motor axon development.

Acknowledgments

We thank Deyou Zheng and Xingyi Guo (Albert Einstein College of Medicine) for assistance with analyses of microarray data; William Andrews (University of London) and Marc Tessier-Lavigne (Genentech) for mutant mice; Minkyung Kim and Brielle Bjorke (University of Nevada) for genotyping; and the following investigators for plasmids: Cathy Krull (University of Michigan; pMES), Hans-Henning Arnold (University of Braunschweig, Germany; mouse Nkx2.9), Marianne Bronner-Fraser (California Institute of Technology; cytopcig-Slit1-LRR), Yi Rao (National Institute of Biological Sciences; Slit2) and Marc Tessier-Lavigne (Robo3 probe). We also thank William Andrews, Jean Hebert, Hannes Buelow, Joseph Locker, Roy Sillitoe and Nozomi Sakai (Albert Einstein College of Medicine) for critical comments on the manuscript. Monoclonal antibodies specific for NF (T. M. Jessell and J. Dodd) and Islet1 (T. M. Jessell and S. Brenner-Morton) were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biology, Iowa City, IA 52242, USA.

Footnotes

Funding

This work was supported by National Institutes of Health (NIH) grants [R56NS038505 and R01NS038505 to Z.K., RO1 NS054740, RR024210 and GM103554 to G.M.]. A.B.-A. was supported by fellowships from the Einstein Medical Scientist Training Program and Society for Neuroscience’s Neuroscience Scholars Program, and an Edward A. and Lucille Kimmel Scholarship. Deposited in PMC for release after 12 months.

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Development is a proud sponsor of the upcoming Santa Cruz Developmental Biology Meeting, which takes place 11-15 August 2018 at the University of California, Santa Cruz . Registration for this meeting is now open!

Meet the preLighters! In the latest interview with our preLights community, the preLights team caught up with James Gagnon, Assistant Professor at the University of Utah, to talk about his research, how science can be made more open, his enthusiasm for the preLights project and the fun sides of being a junior PI.

To investigate which signalling pathways are regulated by nitric oxide during mouth development in Branchiostoma lanceolatum (amphioxus), Filomena Caccavale used a Travelling Fellowship from Development to visit The Oceanographic Observatory in Banyuls-sur-Mer, France, an area with a thriving natural amphioxus population. Read more on her story here.

Where could your research take you? Join Filomena and apply for the next round of Travelling Fellowships from Development by 25 May 2018.